A technology called microarray has been advanced to be developed and used in biological, medical, and pharmaceutical fields since 1990. The microarray is obtained by immobilizing several tens to several tens of thousands of probes onto a substrate made of glass, plastic, or the like and applying a sample (target) labeled with fluorescent molecules or the like to the substrate so as to detect binding reaction between the probe and the sample with fluorescence or the like. The microarrays have a characteristic that makes it possible to perform comprehensive measurement at one time and are expected to be essential to personalized medicine in the future.
The probes to be immobilized onto the substrate include the following types and the microarrays are named based on the types of the probes. That is to say, well-known have been a DNA microarray (DNA chip) obtained by immobilizing DNAs as the probes onto the substrate, a protein microarray obtained by immobilizing proteins as the probes onto the substrate, a tissue microarray obtained by immobilizing a number of small specimens as the probes onto the substrate, a compound microarray obtained by immobilizing a number of low-molecular compounds as the probes onto the substrate, and the like.
Among them, the DNA microarray (hereinafter, referred to as DNA chip) has been put into practical use at the most advanced level. Studies have been performed actively on analyses of genes relating to diseases, and examination and diagnosis by using the genes and some of them have been put into practical use.
Described is the DNA chip as one mode of the microarray in detail below.
The DNA chip is obtained by spotting (immobilizing) DNAs onto the substrate made of glass, resin, or the like in a grid form. The DNAs (probe DNAs) as the probes that can react with the DNA sample to be labeled specifically are spotted on the DNA chip. Optically detectable luminescent or fluorescent mark is added to an unknown DNA sample to be analyzed. The unknown DNA sample to be analyzed is made to flow onto the DNA chip. With this, the DNA sample bonds to the spotted DNA to form a double strand if the unknown DNA sample and the spotted DNA have a complementary relation. Then, all the DNA samples that have not bonded to the probe DNAs are washed out, the DNA samples to be determined that remain on the DNA chip are made luminescent, and the DNA chip is read by a reading device (scanner). This makes it possible to observe the state of the double-stranded DNA as an image. That is to say, distribution of luminescent marks on the DNA chip is analyzed so as to analyze presence of the gene to be obtained, expression of a certain gene, or the degree of expression of the gene. In this manner, an already-known probe DNA set is configured on the DNA chip and the probe DNAs are mounted on a number of types of DNA chips so as to detect genetic alteration, an expression amount of the gene, and the like.
Hereinafter, FIG. 1 illustrates a series of processing processes of DNA chip analysis in detail.
In a preprocessing process as illustrated in FIG. 1, unknown DNA contained in a DNA sample extracted from a specimen is amplified and a fluorescent mark is added to the DNAs.
In the subsequent hybridization process, the DNA sample added with the fluorescent mark (for example, Cy3, Cy5, or the like) are made to drop onto the substrate of the DNA chip on which a number of types of probe DNA have been mounted. The DNA sample bonds to the spotted DNA to form a double strand if the DNA sample and the spotted DNA have a complementary relation.
Next, in the washing process, the hybridized DNA chip is washed with predetermined washer fluid. With this, all the DNA samples that have not bonded to the probe DNAs arranged in the grid form are washed out.
Subsequently, the washed DNA chip is scanned. In the scanning process, the DNA chip is irradiated with a laser beam having a predetermined wavelength suitable for exciting the fluorescent mark (for example, Cy3, Cy5, or the like) so as to be scanned in the reading device. With this, amounts of luminescence of the respective spotted DNAs (genes) are measured and fluorescence image data on which analysis processing is to be performed based on the amounts of luminescence is acquired.
In the analysis process, a fluorescence intensity of each spot is calculated by using a template for the obtained fluorescence image data and various types of analyses are executed.
FIG. 2 illustrates an example of a DNA chip 1 to be used for DNA chip analysis. The DNA chip 1 as illustrated in FIG. 2 includes blocks on a substrate 2. On each of the blocks, a predetermined number of probe DNAs corresponding to individual genes are arrayed in rows and columns in a matrix form (hereinafter, the probe DNA arranged on the block is referred to as a “spot” 3). It is to be noted that the spots 3 arranged on the substrate 2 correspond to genes of which base sequences have been already mapped and that are different from one another and arrangement positions of the spots 3 on the substrate 2 have been defined previously.
FIG. 3 illustrates an example of the template to be applied to the fluorescence image data of the DNA chip. As illustrated in FIG. 3, the template is divided into a plurality of blocks of 1 to 32, for example. Detection areas (corresponding to individual spots of the DNA chip) that are arranged in a matrix form of m rows×n columns (22×22 in FIG. 3) are provided on each block.
In the above-mentioned analysis process, the detection areas on the template provided by an analysis tool are assigned to the individual spots in the fluorescence image data read from a DNA chip (alignment) so as to calculate fluorescence intensities of the respective spots in the corresponding detection areas. In this case, the alignment processing needs to be executed accurately such that the individual detection areas of the template are set correctly to the individual spots on the image in order to execute accurate analysis.
The alignment method includes a pattern matching method and a projection method in which alignment is made on a block basis. As described in Patent Literature 1, alignment tries to be performed accurately by using a chip spotted with a fluorescent substance called positive control or a house-keeping gene contained in any specimens.